13C-13C rotational resonance width distance measurements in uniformly 13C-labeled peptides

The rotational resonance width ((RW)-W-2) experiment is a constant-time version of the rotational resonance (R-2) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R-2 is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine (RW)-W-2 with two-dimensional C-13-C-13 chemical shift correlation spectroscopy and demonstrate the utility of this technique for the site-specific measurement of multiple C-13-C-13 distances in uniformly labeled solids. The dipolar truncation effects, usually associated with distance measurements in uniformly labeled solids, are considerably attenuated in (RW)-W-2 experiments. Thus, (RW)-W-2 experiments are applicable to uniformly labeled biological systems. To validate this statement, multiple C-13-C-13 distances (in the range of 3-6 Angstrom) were determined in N-acetyl-[U-C-13,N-15]L-Val-L-Leu with an average precision of +/-0.5 Angstrom. Furthermore, the distance constraints extracted using a two-spin model agree well with the X-ray crystallographic data.